Human Lactoferrin is an iron-binding single chain polypeptide of 692 amino acids organized into two globular lobes, representing its N-terminal and C-terminal. Each lobe is itself folded into two domains (N-lobe: N1 and N2; C-lobe: C1 and C2) that enclose the iron binding sites. This two-lobe, four-domain structure provides the key to understanding the dynamic properties of lactoferrin. Lactoferrin undergoes a conformational change as iron is bound (closed form) or released (open form). Lactoferrin is a multifunctional glycoprotein produced and secreted by acinar epithelial cells and neutraphils. It is a member of the transferring family of iron binding proteins, but it is also reported to have anti-microbial, antioxidant and immunomodulatory activities. The mature lactoferrin (LF) polypeptide is relatively resistant to proteolysis, is glycosylated at two sites (N138 and N478) and has a molecular weight of about 80 kD.
Lactoferrin is found in the granules of neutrophils where it apparently exerts an anti-microbial activity by withholding iron from ingested bacteria and fungi; it also occurs in many secretions and exudates (milk, tears, mucus, saliva, bile, etc.). In addition to its role in iron transport, lactoferrin has bacteriostatic and bactericidal activities, in addition to playing a role as an antioxidant.
Human milk lysozyme, called muramidase or peptidoglycan N-acetylmuramoyl-hydrolase (EC 3.2.1.17) contains 130 amino acid residues and is a protein of 14.7 kDa in size. Human lysozyme is non-glycosylated and possesses unusual stability in vitro and in vivo due to its amino acid and secondary structure. Lysozymes act as enzymes that cleave peptidoglycans, and ubiquitous cell wall component of microorganisms, in particular bacteria. Specifically, lysozymes are 1,4-β-acetylmuramidases that hydrolyze the glycoside bond between N-acetylmuramic acid and N-acetylglucosamine. Gram-positive bacteria are highly susceptible to lysozyme due to the polypeptidoglycan on the outside of the cell wall. Gram-negative strains have a single polypeptidoglycan layer covered by lipopolysaccharides and are therefore less susceptible to lysis by lysozyme, however, the sensitivity can be increased by the addition of EDTA (Schüitte, H. and Kula, M. R. (1990) Biotechnol. Appl. Biochem. 12: 599–620).
Lactoferrin and lysozyme are found in saliva of healthy individuals, along with IgA, histatins and other natural defense proteins. Saliva flow and anti-microbial proteins appear to function together to maintain oral health by cleansing the mouth of debris and action of oral host defense proteins. The two proteins lactoferrin and lysozyme have anti-fungal activity individually and in combination and their combined action can be synergistic.
Human lactoferrin binds a specific receptor in Caco-2 cells for transport across the membrane. Bovine lactoferrin does not bind this receptor. Human lysozyme has significantly greater lytic activity than the commonly used chicken egg white lysozyme.
Candida albicans is a component found in the mouth of many healthy people. However, oral candidiasis caused by this and related organisms, is one of the most common opportunistic infections of the oral cavity. Oropharyngeal candidiasis (OPC) is a frequent infection in immunocompromised individuals, particularly HIV/AIDS and cancer patients. It is also found associated with steroid drug therapy, diabetes, high carbohydrate diet and other immunosuppressive conditions. The tongue and buccal mucosa lesions, although not considered life threatening, can be extremely uncomfortable and result in decrease food and liquid intake as well as reduced compliance in taking medications. Failure to treat or resolve the oral infection can lead to spread of the infection to the esophagus and ultimately to disseminated candidiasis.
In addition to the difficulties of candidiasis itself, a number of recent reports have dealt with development of resistance to the azoles in patients treated for oropharyngeal candidiasis (OPC). This resistance appears to develop in both chronically and intermittently treated patients. Resistance is more common in immunosupressed patients than otherwise healthy patients. The development of resistance may manifest itself by treatment failure due to replacement of C. albicans with a strain more inherently resistant, such as C. dubliniensis or the development of resistance in C. albicans itself. The infection can be controlled in many resistant strains by increasing the dosage of the azole compounds; however, this can lead to increased toxicity and side effects. The institution of HAART (highly active anti-retroviral treatment) has reduced the frequency of oral Candida albicans infections in HIV and AIDS patients, however, it is not clear whether this is due to the action of the protease inhibitors on aspartic proteinase or the improvement in the immune system with the increase in the number of CD4+T cells. There are also anecdotal reports of recurrent OPC during HAART.
Conventional technology has disclosed methods of producing anti-microbial and anti-fungal protein agents in recombinant hosts, such as monocotyledon plants. These methods, however, produce low quantities of resulting protein product, thereby making these methods inefficient and potentially costly in an economic sense.
U.S. Pat. No. 6,569,831 to Legrand et al (“Legrand”) discloses methods of production of recombinant protein, such as lactoferrin, from plants. Legrand teaches the use of lactoferrin, proteins as anti-microbial agents and anti-fungal agents, particularly against Candida. The method includes transforming a monocot plant cell with a recombinant vector, which comprises a nucleic acid molecule encoding for lactoferrin. Legrand teaches the use of a genetically transformed plant, chosen from amongst rape, tobacco, maize, peas, tomatoes, carrots, wheat, barley, potatoes, soy, sunflower, lettuce, rice, alfalfa, and beets. These genetically transformed plants are manipulated to produce lactoferrin. Legrand teaches the use of several promoters in its recombinant technology. These include 35S (P35S), Pd35S of the CaMV, PCRU of the radish cruciferin, PGA1 and PGA6 of Arabidopsis thalianai, PSP from Agrobacterium tumefaciens, rice actin promoter followed by the actin intron, barley HMWG, and maize γzein gene promoter (Pγzein).
The protein is then expressed in the plant cell and isolated. According to Legrand, the ratio of recombinant human lactoferrin varies by the selection of transformant. According to the methods disclosed in this reference, however, the percent ratio of recombinant human lactoferrin to total soluble proteins in monocot leaves reached 0.1% in the best case. Immunodetection in maize seed was also observed, but no quantities of expression were provided.
The method disclosed in Legrand to produce lactoferrin using genetically transformed monocots provides a general framework for recombinant human protein production in plants. However, Legrand does not disclose a method that produces a relatively high-yield of protein product. For instance, Legrand does not teach a method that can produce protein products greater than 3% of the total soluble protein in the monocot seed.
It would be advantageous to provide a safe, affordable, recombinantly produced anti-fungal agent for treating oral infections in immuno-compromised patients and others. It would also be advantageous to have a method of producing an anti-infective protein agent using recombinant technology in monocots that results in a relatively high yield of the protein product.